Assessment of microbial contamination in laser materials processing laboratories used for prototyping of biomedical devices

Microbial contamination of medical devices during pilot production can be a significant barrier as the laboratory environment is a source of contamination. There is limited information on microbial contaminants in laser laboratories and environments involved in the pilot production of medical devices. This study aimed to determine the bioburden and microbial contaminants present in three laser laboratories – an ISO class 7 clean room, a pilot line facility and a standard laser laboratory. Microbiological air sampling was by passive air sampling using settle plates and the identity of isolates was confirmed by DNA sequencing. Particulate matter was analysed using a portable optical particle counter. Twenty bacterial and 16 fungal genera were isolated, with the genera Staphylococcus and Micrococcus being predominant. Most isolates are associated with skin, mouth, or upper respiratory tract. There was no significant correlation between microbial count and PM2.5 concentration in the three laboratories. There were low levels but diverse microbial population in the laser-processing environments. Pathogenic bacteria such as Acinetobacter baumannii and Candida parapsilosis were isolated in those environments. These results provide data that will be useful for developing a contamination control plan for controlling microbial contamination and facilitating advanced manufacturing of laser-based pilot production of medical devices.


INTRODUCTION
Laser technologies are an important prototyping tool for modifying materials and imbuing them with new properties.Laser technologies are applied in manufacturing various medical devices.Laser cutting has found wide application in manufacturing flexible implantable devices such as stents, catheters, meshes and orthodontic wires [1,2].Furthermore, laser micromachining has enabled high-precision, non-destructive structuring used for producing delicate features on different metal and polymer surfaces using ultrashort pulse lasers.It has been applied in fabricating microneedle drug delivery systems, drug infusion catheters, coronary stents and micro-electrodes [3,4].
Laser structuring often imparts unique properties to materials for potential biomedical applications.For example, using meltfree ultra-short laser femtosecond pulses, our research group have been able to selectively enhance the crystalline structure in molybdenum thin films [5], and improve the conductivity and electrical properties of thin film materials [6][7][8], which could have application in signal enhancement for sensor development.Further, femto-and picosecond lasers have also been used to generate nano-topographical structures on platinum iridium (Pt/Ir) microelectrode probes, leading to improved electrochemical properties and stimulation of key neural functions [9].Contamination of medical devices by micro-organisms compromises the quality of such devices, hence the requirement to assess the risk of contaminants, including monitoring the manufacturing environment and the manufacturing process, to eliminate or reduce the possibility of device-associated infections in animal trials and during subsequent use in humans [10].Microbial contamination of medical devices can arise from raw materials [11], personnel or the manufacturing environment [12].Although terminal sterilization is used for decontaminating medical devices, some terminal sterilization approaches, such as gamma irradiation and ethylene oxide, have limited applications in tissue engineering and regenerative medicine applications due to material incompatibility [13].Hence, the emphasis on contamination control during medical device manufacturing.
While there are efforts to reduce microbial contamination of medical devices, such as redesigning packages and modifying their opening techniques for aseptic transfer [14], the reduction of contamination during production remains a priority.Since the environment is a major source of microbial contamination during manufacturing, and little is known about the levels and types of microbial contaminants in environments where lasers are used to process materials for biomedical prototypes, the following questions are raised.(1) What are the micro-organisms present in typical laser laboratories, which have laser sources, beam delivery systems, motion stages and air-handling systems?(2) What are the levels of these micro-organisms?(3) Are there similarities in the levels and types of micro-organisms in the different laser laboratories?Hence, the aim of this study was to determine the level of microbial contamination, types of microbial contaminants present and whether there are similarities or differences in the microbial contamination in three different laser materials processing laboratories.The results support the development of a contamination control plan for manufacturing laser-enabled regenerative medical products.

Study location
This study was conducted in three laser laboratories, namely the ISO class 7 clean room, a newly refurbished pilot line facility and a standard laser laboratory.The clean room houses a femtosecond laser system, has an H14 high-efficiency particulate air (HEPA)-filtered air exchange system and requires the use of shoe covers, laboratory coat and face coverings as personal protective equipment.The pilot line facility is a newly refurbished laboratory that houses a new multifunctional manufacturing line for pilot production of medical devices.The pilot manufacturing line comprises three separate workstations.Workstation 1 is an ultraviolet laser ablation workstation, workstation 2 is an infrared laser sintering/ablation workstation and workstation 3 is a micro-droplet printing workstation.The pilot line facility requires the use of laboratory coat and face covering.The standard laser laboratory houses a CO 2 laser system and only requires the use of a face covering.Air handling in the pilot line and the standard laser laboratories is controlled by a Mitsubishi Electric CITY MULTI system and consists of 30-40 % air from outside with recycled air from the laboratory.All laboratories have local air extraction from the laser material interaction zone.The laboratories had low occupancy rates, with between one and three individuals in the room while it was being sampled during this study.Further descriptions of the laboratory environment are presented in Table 1.

Microbial air sampling and colony count
The microbiological air sampling was done by passive air sampling using settle plates and was monitored for 5 consecutive weeks September-October 2021.Tryptone soy agar (TSA) and malt extract agar (MEA) plates were used for bacterial and fungal counts, respectively.During sampling, four TSA and MEA plates each were opened and exposed to the air at pre-defined positions in each of the rooms and within the pilot line workstations for 1 h.Each location was divided into four spots to ensure the full coverage of the location.After exposure, the plates were covered, labelled and taken to the incubator.TSA plates were incubated at 37 °C for 48 h and MEA plates were incubated at 30 °C for 5 days.The number of colonies in each plate was counted and recorded and the median colony number for each location determined.The limit of detection (LOD) is 1 c.f.u./plate.

Identification of micro-organisms
Bacterial and fungal isolates were subcultured on TSA and MEA, respectively, and their morphological characteristics recorded.Identification of the isolates was confirmed using DNA-based approaches.16S rDNA gene sequencing was used for bacterial identification using primers 27F (5′-AGAGTTTGATC MTGGCTCAG-3′) and 1492R (5′-TACG GYTA CCTT GTTA CGACTT-3′) [15], while fungi were identified by sequencing the ITS region and large subunit ribosomal RNA genes using the ITS1F (5′-CTTG GTCA TTTA GAGG AAGTAA-3′) and LR3 (5′-CCGTGTTTCAAGACGGG-3′) [16].For bacteria, DNA template was obtained using single colonies resuspended in 250 µl of sterile nuclease-free water.Fungal DNA was extracted using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer's instructions.One microlitre of extracted DNA/colony suspension (DNA template) was used in the PCR using Phusion High-Fidelity DNA Polymerase (Thermo Scientific Fisher, USA).PCR products were purified using a QIAquick Gel Extraction kit (Qiagen) and sequencing was performed by Eurofins Genomics (UK).Sequences were assembled on DNA Baser version 5 (Heracle BioSoft, USA) and the identity of the sequences confirmed by basic local alignment search tool (blast) analysis on the National Center for Biotechnology Information (NCBI) nucleotide database.

Detection of particulate matter
Particulate matter 2.5 (PM 2.5 ) concentrations in the environments were determined using a portable optical particle counter, the SPS30 particulate matter sensor (Sensirion AG, Switzerland).Measurements were performed before operation (at rest) and during operation in the rooms using two independent sensors.PM was measured was measured for 1 h and data collection was repeated on 5 different days.

Statistical analysis
Means ranks of the bacterial and fungal counts, and PM 2.5 in the laboratories and within specific workstations of the pilot manufacturing line were compared using the Kruskal-Wallis test.Non-parametric Spearman correlation was used to determine the relationship between microbial counts and PM 2.5 .All statistical analyses were carried out using GraphPad Prism for Windows version 8.0.2 (GraphPad Prism, RRID:SCR_002798).A P value <0.05 was considered statistically significant.

RESULTS
The air sampling results in laser-processing environments are presented in Table 1.Median bacterial count, at rest, was <LOD in the three laboratories, although the ranges vary in the clean room (<LOD -2 c.f.u./plate), the pilot line facility (<LOD -3 c.f.u./ plate) and the standard laser laboratory (<LOD -4 c.f.u./plate) (Table 2).During operation, there was a higher range of bacterial contamination in the clean room (<LOD -8 c.f.u./plate) and pilot line facility (<LOD -5 c.f.u./plate) compared to the standard laser laboratory (<LOD -2 c.f.u./plate), although the median bacterial count observed in all the laboratories was below the LOD.
Of the three laser materials processing laboratories analysed at rest, the pilot line facility had significantly higher (P=0.0014)median fungal count (1.0 c.f.u./plate) compared to the clean room (<LOD) (Table 2).There was no significant difference (P>0.05) in the fungal count and range among the laboratories during operation.Overall, there was no significant difference in the bacterial and fungal counts at rest and during operation in the three laser materials processing laboratories.
Microbial counts within the workstations of the pilot manufacturing line, located within pilot line facility, were determined.At rest, although the median bacterial count at workstation 2 was 1 c.f.u./plate, there was no significant difference (P>0.05)among them (Table 3).During operation, workstation 2 had a significantly higher (P=0.0472)median bacterial count (1.0 c.f.u./plate) than workstation 1, but it was not significantly different (P>0.05) from that of workstation 3.For fungal count at rest, the median counts in all the workstations were below the LOD (Table 3).Similar to the results from laboratories, there was no significant difference in the bacterial and fungal counts at rest and during operation within the workstations.
Data on the monitoring of the bacterial count in the laser materials processing laboratories over 5 weeks are shown in Fig. 1.
The observed bacterial counts ranged from <LOD -8 c.f.u./plate.The bacterial counts during operation were generally lower than at rest, although there was no significant difference (P>0.05) between them (Fig. 1a, b).There was no significant difference (P>0.05) in the bacterial counts in each location across the weeks except during operation in week 1 (Fig. 1b).During operation in week 1, bacterial count in the clean room (median=5 c.f.u./plate) was significantly higher (P=0.021)than in the pilot line facility (median=2 c.f.u./plate) and the standard laser laboratory (median = <LOD).The fungal counts in the laser materials processing laboratories ranged from <LOD -3 c.f.u./plate.Fungal counts were lower than the bacterial counts both at rest and during operation (Fig. 1c, d).The trend of microbial contamination within the pilot line workstations is shown in Fig. 2.There was no significant difference (P>0.05) in the bacterial and fungal counts in each workstation across the weeks (Fig. 2a).
In this study, 20 bacterial genera were isolated in the laser materials processing laboratories (Fig.

Standard laser laboratory
LOD, limit of detection (1 c.f.u./plate).*Same letter within a column denotes no significant difference (P>0.05) between mean ranks from different environments.† ‡ Different letters within a column denotes a significant difference (P<0.05) between mean ranks from different environments.
Table 3. Microbial counts within the pilot manufacturing line workstations LOD, limit of detection (1 c.f.u./plate).* † ‡-Same letter within a column denotes no significant difference (P>0.05) between means from different workstations and channels.A total of 16 fungal genera were isolated in this study; 13 genera were isolated in the laser materials processing laboratories (Fig. 3) and 9 genera were isolated within the pilot line workstations (Fig. 4).The fungal genera isolated from the laser materials processing  Fungal isolates from workstation 1 had the least diversity among the pilot line workstations, as there were only three fungal isolates, while workstation 3 had five genera ( To determine if there was a relationship between the concentrations of particulate matter and microbial population in the laserprocessing environment, the concentrations of particulate matter (PM 2.5 ) at rest and during operation were determined in the three laser-processing environments.Of the three laser-processing environments at rest, the clean room had the significantly lowest (P<0.0001)PM 2.5 mass concentration (median=0.01µg m −3 ) and number concentration (median=1.29×10 4particles m −3 ), while the pilot line facility had the highest median mass concentration (1.33 µg m −3 ) and median mass concentration (9.87×10 6 particles m −3 ) (Table 4).There was no significant difference (P>0.05) between the mass and number concentrations of the pilot line facility and the standard laser laboratory.The standard laser laboratory was not in use during the PM 2.5 determination, hence no data were collected.Similar to the data obtained at rest, there was a significant difference (P<0.0001) between PM 2.5 mass concentration and number concentration of the pilot line facility and clean room during operation, with the pilot line facility having higher mass-(median=1.22µg m −3 ) and number concentrations (median=8.99×10 6particles m −3 ) than the clean room (median mass concentration=0.03µg m −3 ; median number concentration=9.24×10 4 particles m −3 ).Significantly higher PM 2.5 mass and number concentrations were observed in the clean room during operation than when at rest, unlike in the pilot line facility, where there was no significant difference in the mass and number concentrations at rest and during operation.

Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis and
There was a weak positive correlation between bacterial count and PM 2.5 mass concentration (Spearman r=0.09;P=0.85) and between bacterial count and PM 2.5 number concentration (Spearman r=0.14;P=0.76).Furthermore, there was a weak negative correlation between fungal count and PM 2.5 mass concentration (Spearman r=−0.16;P=0.75) and between bacterial count and PM 2.5 number concentration (Spearman r=−0.05;P=0.93).Overall, there was no significant correlation between microbial count and PM 2.5 concentration in the different laser materials processing laboratories.

DISCUSSION
This study describes microbial contaminants present in laser laboratories and environments involved in the pilot production of biomedical devices.The microbial contamination counts observed in this study were within the recommended limits for the ISO class 7, equivalent to grade C according to the European Union (EU) Good Manufacturing Practice (GMP) [17].No microbial count at any of the weeks exceeded the maximum acceptable levels of index of microbial air contamination in very high-risk environments (5 c.f.u./plate), such as ultra clean rooms [18].To our knowledge, this is the first study of its kind in laser laboratories.The bacterial and fungal counts in this study were also compared to those for a single-occupant office environment and there were higher populations in the office (bacteria=7.33c.f.u./plate; fungi=2.33 c.f.u./plate).Furthermore, the counts observed in the laser-processing environments were lower than those reported in a tissue culture laboratory [19], libraries [20,21], other indoor environments [22,23] and food production environments [24][25][26].Furthermore, the microbial counts found in this study were lower than those reported in other controlled environments, such as operating theatres (at rest=7.2×10 2 c.f.u./plate and during operation=1.05×10 4c.f.u./plate) [27].The authors [27] found a strong significant correlation (Spearman's rank correlation coef-ficient=0.96-0.99) between active (volumetric) air sampling and passive (settle plate) sampling.
In this study, there was no significant difference between the bacterial counts at rest and during operation in the three laser laboratories.This could be because of the use of PPE, particularly facemasks, by the few personnel working in the laboratories as part of coronavirus disease 2019 (COVID-19) mitigation strategies in the workplace during the sampling period [28].Wearing of facemasks reduces the dissemination of aerosols and micro-organisms from the mouth, nose and face into the environment [29].The predominant bacterial genus found in this study was Staphylococcus, with seven species.All seven staphylococcal species observed in this study have been previously found in living rooms [30].Micrococcus yunnanensis and Staphylococcus epidermidis were detected in all three laser-processing environments and they have been isolated in various indoor environments such as homes, classrooms, research laboratories and hospitals [31,32].Micrococcus and Staphylococcus sp., along with Corynebacterium sp., are associated with human skin [33].Many of the isolates obtained during operation in the clean room, such as Rothia, Neisseria and Streptococcus sp., are associated with the oral cavity and upper respiratory tract [34].This suggests that face coverings might not have been used during the operation, hence the need for appropriate use of face coverings during operation in the clean room.
Acinetobacter radioresistens, a radiation-resistant bacterium found on skin, was only isolated in the uncontrolled environments, i.e. the pilot line facility and standard laser laboratory.This bacterium is a source of opportunistic infections and a major source of carbapenem resistance [35].Others, such as Dermacoccus nishinomiyaensis, Staphylococcus capitis and Staphylococcus haemolyticus, are also associated with the skin and can cause infections in humans [36][37][38], hence the need to cover the face and bare skin when working in the laser-processing environments.Most of the isolates unique to both the pilot line facility and standard laser laboratory were predominantly environmental organisms related to the external environment, particularly the soil.This indicates the importance of wearing appropriate PPE, such as shoe coverings, in uncontrolled laser-processing environments in order to reduce microbial contamination.While the micro-organisms in the pilot line workstations shared some similarities with the pilot line facility, in which they are located, there were still some isolates found in the workstations but not in the pilot line facility.For example, Acinetobacter baumannii was isolated in workstation 3, which raises a huge health concern, as it is an important pathogen capable of causing multiple infections in humans [39], particularly among the immunocompromised and critically ill patients.Environmental A. baumannii isolates have been reported to harbour multiple drug-resistant genes [40] and carbapenem-resistant A. baumannii has been identified to be of critical priority for developing new antibiotics [41].
The only fungus isolated in the clean room was Candida parapsilosis, which is a commensal of humans but is also isolated in soil.This fungus poses a significant health threat because of its role in invasive infections such as bloodstream infections [42].Furthermore, C. parapsilosis has been identified as an emerging antifungal-resistant organism worldwide [43].Penicillium sp. and Candida intermedia were found in the pilot line facility and standard laser laboratory.Penicillium sp. is a common fungus found in indoor and outdoor environments, and its indoor levels have been associated with asthma morbidity [44].On the other hand, C. intermedia, which predominantly occurs on human skin and in the throat but also in soil, can cause blood infections in humans [45].
The significantly higher fungal count observed in the pilot line facility in this study could be due to the laboratory's proximity to the general corridor and the fact that no footwear covering is worn, which may transfer fungi from outdoor environments into the laboratory.It has been reported that fungi in indoor air are dominated by fungi from outdoor air [46].Conversely, the significantly lower fungal counts found in the clean room could be due to the clean room being further away from the corridor and having a changing room where laboratory coats and shoe coverings are worn prior to entry.Further, the presence of air filtration/ventilation systems, such as HEPA filters, in the clean room may also reduce fungal counts [47].Continuous ventilation is recommended for reducing finer particulate matter (<30 nm) in indoor environments as it reduces mass concentration of particulate matter faster than when motion sensor-regulated ventilators are used [48].This point could be further corroborated by the fact that only one fungal isolate was detected in the clean room, which is a controlled environment, compared to the pilot line facility and standard laser laboratory, which are uncontrolled environments.Since some of the fungal isolates in the indoor laser-processing environments, particularly the uncontrolled environments, have also previously been reported in outdoor environments, they may have originated from the outdoor air [49].
The mass concentration of PM 2.5 (range=0.00-3.76µg m −3 ) detected in this study were below the EU air quality standard limit of 20 µg m −3 for PM 2.5 [50].The low particle numbers in this study could be because of the requirement of PPE use based on the COVID-19 pandemic guidelines for working in laboratories.Similar to this study, Landrin et al. [51] found no significant correlation between particle counting and microbiological sampling, although correlations between bacterial counts by passive sampling and PM 2.5 in hospital environments have been reported [52].
In conclusion, this study showed that there is a low level of microbial contamination in laser-processing environments.Twenty bacterial and 16 fungal genera were isolated in this study, with the pilot line facility having the highest microbial diversity (13 bacterial and 11 fungal genera).Most of the isolates are associated with skin, mouth and upper respiratory tract, but some are potentially pathogenic.Staphylococcus is the most common and most diverse genus isolated.Finally, there was no significant correlation between the microbial count and PM 2.5 concentration in the laser-processing environments.These results provide data that will be useful for developing a contamination control plan for controlling microbial contamination and facilitating advanced manufacturing of laser-based pilot production of medical devices.Based on our results, it is recommended that shoe covers and facemasks be worn in laboratories used for prototyping medical devices to minimize microbial contamination.The Microbiology Society is a membership charity and not-for-profit publisher.
Your submissions to our titles support the community -ensuring that we continue to provide events, grants and professional development for microbiologists at all career stages.Comments: This manuscript details an interesting study investigating the level of microbial contamination in three laser processing laboratories, in addition to providing genotypic identification for the organisms recovered.The manuscript also samples the air quality during use and at rest for the locations.In general, the article is well written and informative, and of an appropriate length.The number of figures and tables is suitable for the data presented.The title of the article makes reference to 'biomedical concepts' -this is somewhat vague and could be amended to 'biomedical devices' or similar.The Introduction specifies that the laser materials processing laboratories are involved in the production of medical devices, materials fabrication and machining, and improving electrical properties of materials for biomedical sensing.The research need is clearly established, however elaboration on the reasons why terminal sterilisation methods have limited application would be welcomed.The aims are well set out, but make reference to a 'typical' laser laboratory, which is not immediately obvious to non-specialists.The Methods section is generally very good.The descriptions of the different laboratories and workstations are helpful and welcomed.The manuscript states the occupancy levels for the various locations, but including an estimate of the length of time per 'visit' would help to contextualise this.The investigative approach has no major flaws or errors, but some omissions are noted.For example, number of settle plates used, number of replicates, and details regarding the 'pre-defined' positions of the (detailed in major comments below).After recovering microorganisms from the environment, standard genomic techniques were used to identify them -it would be welcomed if the primers detailed for this were appropriately cited.The findings were significant as it provided data that will be used in future to control contamination in such laboratories.Findings offer advancement in knowledge for it specify particular microorganisms which can still be found in such controlled environments.Results were properly interpreted and were based on sound data.3. How the style and organization of the paper communicates and represents key findings Introduction uses literature to connect problem statement and purpose of the study.The study gives clear and measurable objectives on which the design and method is based.The method of data collection was precise and reproducible and gave sound data.The data analysis method was adequate for the type of data collected.Inference from the data clearly specified key findings.4. Literature analysis or discussion Literature review was adequate.The review in introduction was well directed to both problem statement and purpose of the study.However in discussion there were no mention of similar studies in similar environment probably this was the first study of its kind in laser laboratories.5. Any other relevant comments I think it could have been important to mention, in discussion, that this is the first study of its kind in laser laboratories after a thorough such of literature for similar studies without success.

Please rate the manuscript for methodological rigour Very good
Please rate the quality of the presentation and structure of the manuscript

Very good
To what extent are the conclusions supported by the data?Strongly support

Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices? No
Is there a potential financial or other conflict of interest between yourself and the author(s)?No If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Staphylococcus saprophyticus.Micrococcus yunnanensis and S. epidermidis were detected in all three laser-processing environments.Acinetobacter radioresistens and Dermacoccus nishinomiyaensis were isolated in the standard laser and the pilot line facility but not in the clean room.Paracoccus yeei, Neisseria sp., Rothia dentocariosa, Streptococcus cristatus and Streptococcus mitis were only detected in clean room.Niallia circulans, Ornithinibacillus bavariensis, Pantoea sp. and S. caprae were only detected in the standard laser laboratory, while Arthrobacter

Fig. 2 .
Fig. 2. Trend of microbial contaminants within workstations in a pilot manufacturing line: Bacterial count at rest (a) and during operation (b) as well as fungal count at rest (c) and during operation (d), with four replicates at each location.Error bars represent 95 % confidence interval (CI) of median.

Fig. 1 .
Fig. 1.Trend of microbial contaminants in different laser materials processing laboratories.Bacterial count at rest (a) and during operation (b) as well as fungal count at rest (c) and during operation (d), with four replicates at each location.Error bars represent 95 % confidence interval (CI) of median.

Table 1 .
Description of the laser materials processing environments

Table 2 .
Microbial counts in different laser materials processing laboratories Kocuria and Mesobacillus were found in the pilot line facility but not in other laser materials processing laboratories.M. yunnanensis and D. nishinomiyaensis were found in all the workstations.S. hominis and S. capitis were detected in all workstations.S. epidermidis was detected in all workstations.Microbacterium oxydans was only found in workstations 1 and 2, whereas Micrococcus aloeverae and S. haemolyticus were detected in workstations 2 and 3. Bacillus subtilis, Curtobacterium oceanosedimentum, Kocuria sp., Paenibacillus provencensis, Staphylococcus petrasii and Staphylococcus warneri were only detected in workstation 1.The isolates found solely in workstation 2 were Paracoccus yeei, Brachybacterium sp., Mesobacillus and Micrococcus luteus.A. baumannii, Moraxella osloensis and S. saprophyticus were only detected in workstation 3. Isolates detected within the pilot line workstations but not found in the pilot line facility include A. baumannii, Bacillus simplex, Brachybacterium sp., C. oceanosedimentum, Kocuria sp., Mesobacillus sp., M. oxydans, M. osloensis, P. provencensis, P. yeei, S. petrasii and S. warneri.

Table 4 )
. Peniophora sp. and Lecanicillium dimorphum were only detected in workstation 1; Coprinellus sp. and Penicillium sp. were only detected in workstation 2, while Zopfiella marina and Mycoacia fuscoatra were only detected in workstation 3. Agaricomycetes sp., C. intermedia, Coprinellus sp., Lecanicillium sp., Penicillium sp. and Peniophora sp. were detected both within the pilot line workstations and in the pilot line facility.However, M. fuscoatra, Trametes versicolor and Z. marina were found in the workstations but not in the pilot line facility.Furthermore, C. parapsilosis, Sordariomycetes sp., Antrodia sp., Cladosporium sp., Cordyceps sp., P. trinitatensis and T. verruculosus were found in the pilot line facility but not within the pilot line workstations.

Table 4 .
Concentration of particulate matter (PM * †-Different letters in a row denotes a significant difference (P<0.05) between mean ranks from different environments.
and innovation programme under the Marie Sklodowska-Curie grant agreement no.847 652 and from Science Foundation Ireland (12/RC/2276).The research is also motivated by Science Foundation Ireland (grant no.19/US-C2C/3672) and the SFI Research Centre for Medical Devices (grant no.13/ RC/2073_P2).

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The work presented is clear and the arguments well formed.This study would be a valuable contribution to the existing literature.The reviewers have highlighted minor concerns with the work presented.Please ensure that you address their comments.The manuscript (ACMI-D-22-00146) entitled "Assessment of microbial contamination in laser materials processing laboratories used for prototyping of biomedical concepts" by Somorin & O'Connor investigated the presence of pathogens in three medical device production units.They have isolated various bacteria and fungi in different production units demanding attention for future planning to prevent contamination.This study provides important data to the scientific community involved in the production of medical devices as well as the user.The author's way of presentation (Text and graphic) is very understandable to the reader.I have no further comments.
(Daneshnia et al. 2023ts described well.Statistical tests seem appropriate.The Results section is clear and informative, and clearly relates to the methods section.The main findings are presented concisely and effectively.The microbial identification data are nicely presented in the figures and the use of the overlapping coloured circles is welcomed.However the use of Kruskal-Wallis and Spearman's rank tests imply non-normal distribution of the data.If so, median values should be stated throughout instead of mean values.If the data were normally distributed, appropriate parametric equivalent tests should be performed instead to improve power.All figure legends should include the number of replicates and an indication as to what the error bars represent.The Discussion is well presented in general and places the work in a more medical microbiology context, focussing on the sources of contamination for various organisms, as well as citing literature related to potential consequences of contamination for specific organisms.Reference to other microbially controlled locations such as food production facilities and operating theatres provide context and are welcome.In highlighting Candida parapsilosis, its recent status as an emerging antifungal resistant organism should be mentioned(Daneshnia et al. 2023The Lancet, DOI:https:// doi.org/ 10. 1016/ S2666-5247(23) 00067-8).The manuscript gives suggestions for a contamination control plan, which is welcomed -this should also be stated in the abstract as a key outcome.Major comments: Multiple lines: Use of KW and Spearman's tests imply non-parametric datasets.If this is the case, mean values should be replaced with median values throughout.If datasets were normally distributed, appropriate parametric tests should be used instead.Multiple lines: All figure legends must contain number of replicates and an indication of what the error bars represent.Line 122-123: No details on numbers of plates used in each location.No details about location of settle plates or rationale behind these locations.Please provide these details.Minor comments: Line 2: Consider amending 'biomedical concepts' to something more specific such as 'biomedical devices'.Abstract: Please include key outcomes such as identification of major human pathogens (Acinetobacter baumanii, Candida paraspilosis, etc.) and include a short summary of the developed contamination control plan.Line 65: Please make explicit that this was previous work by the group.Line 79-82: Please provide an explanation as to why terminal sterilisation by gamma irradiation or ethylene oxide aren't appropriate, and add a relevant citation.Line 91: Please provide a brief description of a typical laser laboratory.Line 115: An indication into typical visit times to each location would be welcomed.Line 121: TSA and MEA plates are used, but if it were suspected that Streptococcus spp were present then a blood-enriched agar type could've been used.It is unclear if greater numbers of this genera would've then been isolated/recovered, and this should be mentioned in the discussion.Line 133-134 and 136: If the primers detailed were designed by the authors, please detail this in the methods section.Alternatively, please provide a citation for all primers used.Line 235: Full stop missing after 'laboratories'.Please include this.Line 284 and 285: Please state the p-value for the Spearman's correlations in addition to the r-value if possible.Line 346: Please highlight Candida parapsilosis as an emerging antifungal resistant organism.Please rate the manuscript for methodological rigour GoodPlease rate the quality of the presentation and structure of the manuscript Very goodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000494.v1.3 © 2022 Auma A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Methodological rigour, reproducibility and availability of underlying data The objectives were clearly stated.The objectives were measurable and all were met.The methods used for colony counts is reproducible.Identification of microorganisms was confirmed by molecular method.Statistical methods were adequate for the data.2. Presentation of results